A. Field of the Invention
The present invention relates to the field of enzymes. More specifically, it concerns cyanobacterial and plant acetyl-CoA carboxylase (ACC) compositions and methods for making and using ACC-encoding polynucleotides and ACC polypeptides.
B. Description of the Related Art
1. Acetyl-CoA Carboxylase
Acetyl-CoA carboxylase (ACC) catalyzes the first committed step in de novo fatty acid biosynthesis, the addition of CO.sub.2 to acetyl-CoA to yield malonyl-CoA. It belongs to a group of carboxylases that use biotin as cofactor and bicarbonate as a source of the carboxyl group. ACC catalyzes the addition of CO.sub.2 to acetyl-CoA to yield malonyl-CoA in two steps as shown below. EQU BCCP+ATP+HCO.sub.3 --.fwdarw.BCCP--CO.sub.2 +ADP+P.sub.i (1) EQU BCCP--CO.sub.2 +Acetyl-CoA.fwdarw.BCCP+malonyl-CoA (2)
First, biotin becomes carboxylated at the expense of ATP. The carboxyl group is then transferred to Ac-CoA (Knowles, 1989). This irreversible reaction is the committed step in fatty acid synthesis and is a target for multiple regulatory mechanisms. Reaction (1) is catalyzed by biotin carboxylase (BC); reaction (2) by transcarboxylase (TC); BCCP=biotin carboxyl carrier protein.
There are two types of ACC: prokaryotic ACC in which the three functional domains: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP) and carboxyltransferase (CT) are located on separable subunits (e.g., E. coli, P. aeruginosa, Anabaena, Synechococcus and probably pea chloroplast) and eukaryotic ACC in which all the domains are located on one large polypeptide (e.g., rat, chicken, yeast, diatom and wheat).
E. coli ACC consists of a dimer of 49-kDa BC monomers, a dimer of 17-kDa BCCP monomers and a CT tetramer containing two each of 33-kDa and 35-kDa subunits. The primary structures of all of the E. coli ACC subunits (Alix, 1989; Muramatsu and Mizuno, 1989; Kondo et al., 1991; Li and Cronan, 1992; Li and Cronan, 1992) as well as the structure of the BC and BCCP of Anabaena 7120 (Gornicki et al., 1993), and P. aeruginosa (Best and Knauf, 1993) are known, based on the gene sequences. The genes encoding the subunits of E. coli ACC are called: accA (CT a subunit), accB (BCCP), accC (BC) and accD (CT .beta. subunit). accC and accB form one operon, while accA and accD are not linked to each other or to accCB (Li and Cronan, 1992). In cyanobacteria, accC and accB are unlinked as well (Gornicki et al., 1993).
Yeast, rat, chicken and human ACCs are cytoplasmic enzymes consisting of 250- to 280-kDa subunits while diatom ACC is most likely a chloroplast enzyme consisting of 230-kDa subunits. Their primary structure has been deduced from cDNA sequences (Al-feel et al., 1992; Lopez-Casillas et al., 1988; Takai et al., 1988; Roessler and Ohlrogge, 1993; Ha et al., 1994). In eukaryotes, homologs of the four bacterial genes are fused in the following order: accC, accB, accD and accA. Animal ACC activity varies with the rate of fatty acid synthesis or energy requirements in different nutritional, hormonal and developmental states. In the rat, ACC mRNA is transcribed using different promoters in different tissues and can be regulated by alternative splicing. The rat enzyme activity is also allosterically regulated by a number of metabolites and by reversible phosphorylation (Ha et al., 1994 and references therein). The expression of the yeast gene was shown to be coordinated with phospholipid metabolism (Chirala, 1992; Haslacher et al., 1993).
Much less is known relating to plant ACC. Early attempts at characterization of plant ACC led to the suggestion that it consisted of low molecular weight subunits similar to those of bacteria (Harwood, 1988). More recent efforts indicate that at least one plant isozyme is composed of &gt;200-kDa subunits, similar to the enzyme from other eukaryotes (Egin-Buhler and Ebel, 1983; Slabas and Hellyer, 1985; Gornicki and Haselkorn, 1993; Egli et al., 1993; Betty et al., 1992).
While strong evolutionary conservation exists among biotin carboxylases and biotin carboxylase domains of all biotin-dependent carboxylases, BCCP domains show very little conservation outside the conserved sequence E(A/V)MKM (lysine residue is biotinylated) (Knowles, 1989; Samols et al., 1988). Although the three functional domains of the E. coli ACC are located on separate polypeptides, plant ACC is quite different, having all 3 domains on a single polypeptide.
At least one form of plant ACC is located in plastids, the primary site of fatty acid synthesis. The gene encoding it, however, must be nuclear because no corresponding sequence has been seen in the complete chloroplast DNA sequences of tobacco, liverwort or rice. The idea that in some plants plastid ACC consisted of several smaller subunits was revived by the discovery of an accD homolog in some chloroplast genomes (Li and Cronan, 1992). Indeed, it has been shown that the product of this gene in pea binds two other peptides, one of which is biotinylated. The complex may be a chloroplast isoform of ACC in pea and some other plants (Sasaki et al., 1993).
2. Genes Encoding ACC
Plant genes encoding eukaryotic-type plastid ACC are nuclear-encoded, since no corresponding sequences have been identified in the complete chloroplast DNA sequences of tobacco, liverwort or rice. ACC, like the vast majority of chloroplast proteins which are encoded in nuclear DNA, most probably is synthesized in the cytoplasm and then transported into the chloroplast, presumably requiring a chloroplast transport sequence. Although the basic features of plant ACC are the same as those of-prokaryotic and other eucaryotic ACCs, significant differences may also be expected due, for example, to differences in plant cell metabolism and ACC cellular localization.
The possibility of different ACC isoforms, one present in plastids and another in the cytoplasm, is now accepted. The rationale behind the search for a cytoplasmic ACC isoform is the requirement for malonyl-CoA in this cellular compartment, where it is used in fatty acid elongation and synthesis of secondary metabolites. Indeed, two isoforms were found in maize, both consisting of &gt;200-kDa subunits but differing in size, herbicide sensitivity and immunological properties. The major form was found to be located in mesophyll chloroplasts. It is also the major ACC in the endosperm and in embryos (Egli et al., 1993).
3. Herbicide Resistance
Although the mechanisms of inhibition and resistance are unknown (Lichtenthaler, 1990), it has been shown that aryloxyphenoxypropionates and cyclohexane-1,3-dione derivatives, powerful herbicides effective against monocot weeds, inhibit fatty acid biosynthesis in sensitive plants.
The aryloxyphenoxypropionate class comprises derivatives of aryloxyphenoxypropionic acid such as diclofop, fenoxaprop, fluazifop, haloxyfop, propaquizafop and quizalofop. Several derivatives of cyclohexane-1,3-dione are also important post-emergence herbicides which also selectively inhibit monocot plants. This group comprises such compounds as oxydim, cycloxydim, clethodim, sethoxydim, and tralkoxydim.
Recently it has been determined that ACC is the target enzyme for both of these classes of herbicide at least in monocots. Dicotyledonous plants, on the other hand, such as soybean rape, sunflower, tobacco, canola, bean, tomato, potato, lettuce, spinach, carrot, alfalfa and cotton are resistant to these compounds, as are other eukaryotes and prokaryotes.
Important grain crops, such as wheat, rice, maize, barley, rye, and oats, however, are monocotyledonous plants, and are therefore sensitive to these herbicides. Thus herbicides of the aryloxyphenoxypropionate and cyclohexane-1,3-dione groups are not useful in the agriculture of these important grain crops owing to the inactivation of monocot ACC by such chemicals.
4. Cyanobacteria
Unlike monocot plants, members of the cyanobacteria are resistant to these herbicide families. Cyanobacteria are prokaryotes that carry out green plant photosynthesis, evolving O.sub.2 in the light. They are believed to be the evolutionary ancestors of chloroplasts. Virtually nothing is known about fatty acid biosynthesis in cyanobacteria.
Synechococcus is a unicellular obligate phototroph with an efficient DNA transformation system. Replicating vectors based on endogenous plasmids are available, and selectable markers include resistance to kanamycin, chloramphenicol, streptomycin and the PSII inhibitors diuron and atrazine. Inactivation and/or deletion of Synechococcus genes by transformation with suitable cloned material interrupted by resistance cassettes is well known in the art. Genes may also be replaced by specifically mutated versions using selection for closely linked resistance cassettes.
Anabaena differentiates specialized cells for nitrogen fixation when the culture is deprived of a source of combined nitrogen. The differentiated cells have a unique glycolipid envelope containing C26 and C28 fatty acids (Murata and Nishida, 1987), whose synthesis must start with the reaction catalyzed by ACC. Therefore ACC must be developmentally regulated in Anabaena. Powerful systems of genetic analysis exist for Anabaena as well (Golden et al., 1987).
That cyanobacteria and plants are evolutionarily-related make the former useful sources of cloned genes for the isolation of plant cDNAs. This method is well known to those of skill in the art. For example, the cloned gene for the enzyme phytoene desaturase, which functions in the synthesis of carotenoids, isolated from cyanobacteria was used as a probe to isolate the cDNA for that gene from tomato (Pecker et al., 1992).
5. Deficiencies in the Prior Art
The genetic transformation of important commercial monocotyledonous agriculture crops with DNA segments encoding herbicide-resistant ACC enzymes would be a revolution in the farming of such grains as wheat, rice, maize, barley, rye, and oats. Moreover the availability for modulating the herbicide resistance of plants through the alteration of ACC-encoding DNA segments and the polypeptides themselves would be highly desirable. Methods of identifying and assaying the levels of ACC activity in these plants would also be important in genetically engineering grain crops and the like with desirable herbicide-resistant qualities. Likewise the availability of DNA segments encoding dicotyledonous ACC and nucleic acid segments derived therefrom would provide a much-needed means of genetically altering the activity of ACC in vivo and in vitro.
What is lacking in the prior art, therefore, is the identification of DNA segments encoding plant and cyanobacterial ACC enzymes, and the development of methods and processes for their use in creation of modified, transgenic plants which have altered herbicide resistance. Moreover, novel methods providing transgenic plants using DNA segments encoding ACC polypeptides to modulate ACC activity, fatty acid biosynthesis in general, and oil content of plant cells in specific, are greatly needed to provide transformed plants altered in such activity. Methods for determining ACC activity in vivo and quantitating herbicide resistance in plants would also represent major improvements over the current state of the art.